[Corrigendum] Evaluation of eight reference genes for quantitative polymerase chain reaction analysis in human T lymphocytes co­cultured with mesenchymal stem cells.

Following the publication of the above article, the authors contacted the Editorial Office to explain that Fig. 1A and some of the images in Fig. 1B in the paper had already been published in Fig. 1 in another article by the same authors, and they had forgotten to cite the former publication. The paper in which these data appeared was as follows: Li X, Yang Q, Bai J, Xuan Y and Wang Y: Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real­time PCR. Biotechnol Lett 37: 67­73, 2015. Fig. 1 of the above paper is reprinted opposite, now with the original source of the figure acknowledged in the form of a reference citation at the end of the Figure caption. The authors apologize to the publishers of Biotechnology Letters for having failed to include a proper acknowledgement for use of the figure in the above publication. [the original article was published in Molecular Medicine Reports 12: 7721-7727, 2015; DOI: 10.3892/mmr.2015.4396].

A 4-gene signature from histologically normal surgical margins predicts local recurrence in patients with oral carcinoma: clinical validation.

Prognostic biomarkers for recurrence of Oral Squamous Cell Carcinoma (OSCC) are urgently needed. We aimed to independently validate a 4-gene expression signature (MMP1, COL4A1, P4HA2, THBS2) predictive of OSCC recurrence risk. Gene expression was measured using Nanostring nCounter® in 245 histologically normal surgical resection margins from 62 patients. Association between risk scores for individual patients and recurrence was assessed by Kaplan-Meier analysis. Signature performance was quantified by concordance index (CI), hazard ratio (HR) and the area under receiver operating characteristics (AUC). Risk scores for recurrence were significantly higher than recurrence-free patients (p = 9.58e-7, Welch's t-test). A solid performance of the 4-gene signature was determined: CI = 0.64, HR = 3.38 (p = 1.4E-4; log-rank test), AUC = 0.71. We showed that three margins per patient are sufficient to preserve predictive performance (CI = 0.65; HR = 2.92; p = 2.94e-3; AUC = 0.71). Association between the predicted risk scores and recurrence was assessed and showed HR = 2.44 (p = 9.6E-3; log-rank test, N = 62). Signature performance analysis was repeated using an optimized threshold (70th percentile of risks), resulting in HR = 3.38 (p = 1.4E-4; log-rank test, N = 62). The 4-gene signature was validated as predictive of recurrence risk in an independent cohort of patients with resected OSCC and histologically negative margins, and is potentially applicable for clinical decision making on adjuvant treatment and disease monitoring.

Evaluation of eight reference genes for quantitative polymerase chain reaction analysis in human T lymphocytes co­cultured with mesenchymal stem cells.

Accurate gene expression analysis relies on the selection of a stable reference gene, as unstable reference genes can alter experimental results and conclusions. It is widely­accepted that reference genes exhibit different expression levels in different types of tissues and cells. Therefore, it is essential to screen for stably­expressed reference genes in the cells and tissues used for experimental analysis prior to performing reverse transcription­quantitative polymerase chain reaction (RT­qPCR). In the present study, eight reference genes were screened for their suitability for RT­qPCR in five T lymphocytes co­cultured with mesenchymal stem cells from different sources. Using NormFinder, geNorm, and BestKeeper algorithms consistently demonstrated that RPL13A and B2M were the optimal reference genes for the normalization of RT­qPCR data obtained from T lymphocytes, whereas glyceraldehyde 3­phosphate dehydrogenase was not a suitable reference gene due to its extensive variability in expression. These findings highlight the importance of evaluating reference genes for RT­qPCR.

Identification of appropriate reference genes for human mesenchymal stem cell analysis by quantitative real-time PCR.

Normalization to a reference gene is the method of choice for quantitative reverse transcription-PCR (RT-qPCR) analysis. The stability of reference genes is critical for accurate experimental results and conclusions. We have evaluated the expression stability of eight commonly used reference genes found in four different human mesenchymal stem cells (MSC). Using geNorm, NormFinder and BestKeeper algorithms, we show that beta-2-microglobulin and peptidyl-prolylisomerase A were the optimal reference genes for normalizing RT-qPCR data obtained from MSC, whereas the TATA box binding protein was not suitable due to its extensive variability in expression. Our findings emphasize the significance of validating reference genes for qPCR analyses. We offer a short list of reference genes to use for normalization and recommend some commercially-available software programs as a rapid approach to validate reference genes. We also demonstrate that the two reference genes, ß-actin and glyceraldehyde-3-phosphate dehydrogenase, are frequently used are not always successful in many cases.

Comprehensive characterization of four different populations of human mesenchymal stem cells as regards their immune properties, proliferation and differentiation.

In the present study, we compared mesenchymal stem cells (MSCs) derived from 4 different sources, human bone marrow (BM), adipose tissue (AT), umbilical cord Wharton's Jelly (WJ) and the placenta (PL), in order to determine which population of MSCs displayed the most prominent immunosuppressive effects on phytohemagglutinin-induced T cell proliferation, and which one had the highest proliferative and differentiation potential. MSC and T lymphocyte co-culture (mixed culture) was used to determine whether the MSCs inhibit T cell proliferation, as well as which population of MSCs has the strongest inhibitory ability. The expression of immune-related genes was analyzed by RT-PCR and RT-qPCR. The proliferation and differentiation potential of the MSCs were determined using standard methods. Following MSC and T cell co-culture, mitogen-induced T cell proliferation was effectively suppressed by all 4 populations of MSCs. This occurred through soluble factors rather than direct contact inhibition. Among the 4 populations of MSCs, the WJ-MSC has the strongest suppression effects. On immune related genes, WJ-MSC has the weakest expression of MHC II genes, TLR4, TLR3, JAG1, NOTCH2 and NOTCH3. To compare the proliferation potential, WJ-MSCs showed the most rapid growth rate followed by the AT-, PL- and BM-MSCs. As regards differentiation potential, the WJ-MSCs had the strongest osteogenetic ability followed by PL, AT and BM-MSC. AT-MSC has the strongest adipogenetic ability followed by the WJ-, BM- and PL-MSCs. These data indicated that the WJ-MSCs had the strongest immunomodulatory and immunosuppressive potential. In light of these observations, we suggest that WJ-MSCs are the most attractive cell population for use in immune cellular therapy when immunosuppressive action is required.

MicroRNA signature obtained from the comparison of aggressive with indolent non-Hodgkin lymphomas: potential prognostic value in mantle-cell lymphoma.

PURPOSE: Mantle-cell lymphoma (MCL) has a variable natural history but is incurable with current therapies. MicroRNAs (miRs) are useful in prognostic assessment of cancer. We determined an miR signature defining aggressiveness in B-cell non-Hodgkin lymphomas (NHL) and assessed whether this signature aids in MCL prognosis. METHODS: We assessed miR expression in a training set of 43 NHL cases. The miR signature was validated in 44 additional cases and examined on a training set of 119 MCL cases from four institutions in Canada. miRs significantly associated with overall survival were examined in an independent cohort of 114 MCL cases to determine association with patient outcome. miR expression was combined with current clinical prognostic factors to develop an enhanced prognostic model in patients with MCL. RESULTS: Fourteen miRs were differentially expressed between aggressive and indolent NHL; 11 of 14 were validated in an independent set of NHL (excluding MCL). miR-127-3p and miR-615-3p were significantly associated with overall survival in the MCL training set. Their expression was validated in an independent MCL patient set. In comparison with Ki-67, expression of these miRs was more significantly associated with overall survival among patients with MCL. miR-127-3p was combined with Ki-67 to create a new prognostic model for MCL. A similar model was created with miR-615-3p and Mantle Cell Lymphoma International Prognostic Index scores. CONCLUSION: Eleven miRs are differentially expressed between aggressive and indolent NHL. Two novel miRs were associated with overall survival in MCL and were combined with clinical prognostic models to generate novel prognostic data for patients with MCL.

A gene signature in histologically normal surgical margins is predictive of oral carcinoma recurrence.

BACKGROUND: Oral Squamous Cell Carcinoma (OSCC) is a major cause of cancer death worldwide, which is mainly due to recurrence leading to treatment failure and patient death. Histological status of surgical margins is a currently available assessment for recurrence risk in OSCC; however histological status does not predict recurrence, even in patients with histologically negative margins. Therefore, molecular analysis of histologically normal resection margins and the corresponding OSCC may aid in identifying a gene signature predictive of recurrence. METHODS: We used a meta-analysis of 199 samples (OSCCs and normal oral tissues) from five public microarray datasets, in addition to our microarray analysis of 96 OSCCs and histologically normal margins from 24 patients, to train a gene signature for recurrence. Validation was performed by quantitative real-time PCR using 136 samples from an independent cohort of 30 patients. RESULTS: We identified 138 significantly over-expressed genes (> 2-fold, false discovery rate of 0.01) in OSCC. By penalized likelihood Cox regression, we identified a 4-gene signature with prognostic value for recurrence in our training set. This signature comprised the invasion-related genes MMP1, COL4A1, P4HA2, and THBS2. Over-expression of this 4-gene signature in histologically normal margins was associated with recurrence in our training cohort (p = 0.0003, logrank test) and in our independent validation cohort (p = 0.04, HR = 6.8, logrank test). CONCLUSION: Gene expression alterations occur in histologically normal margins in OSCC. Over-expression of the 4-gene signature in histologically normal surgical margins was validated and highly predictive of recurrence in an independent patient cohort. Our findings may be applied to develop a molecular test, which would be clinically useful to help predict which patients are at a higher risk of local recurrence.

mRNA transcript quantification in archival samples using multiplexed, color-coded probes.

BACKGROUND: A recently developed probe-based technology, the NanoString nCounter™ gene expression system, has been shown to allow accurate mRNA transcript quantification using low amounts of total RNA. We assessed the ability of this technology for mRNA expression quantification in archived formalin-fixed, paraffin-embedded (FFPE) oral carcinoma samples. RESULTS: We measured the mRNA transcript abundance of 20 genes (COL3A1, COL4A1, COL5A1, COL5A2, CTHRC1, CXCL1, CXCL13, MMP1, P4HA2, PDPN, PLOD2, POSTN, SDHA, SERPINE1, SERPINE2, SERPINH1, THBS2, TNC, GAPDH, RPS18) in 38 samples (19 paired fresh-frozen and FFPE oral carcinoma tissues, archived from 1997-2008) by both NanoString and SYBR Green I fluorescent dye-based quantitative real-time PCR (RQ-PCR). We compared gene expression data obtained by NanoString vs. RQ-PCR in both fresh-frozen and FFPE samples. Fresh-frozen samples showed a good overall Pearson correlation of 0.78, and FFPE samples showed a lower overall correlation coefficient of 0.59, which is likely due to sample quality. We found a higher correlation coefficient between fresh-frozen and FFPE samples analyzed by NanoString (r = 0.90) compared to fresh-frozen and FFPE samples analyzed by RQ-PCR (r = 0.50). In addition, NanoString data showed a higher mean correlation (r = 0.94) between individual fresh-frozen and FFPE sample pairs compared to RQ-PCR (r = 0.53). CONCLUSIONS: Based on our results, we conclude that both technologies are useful for gene expression quantification in fresh-frozen or FFPE tissues; however, the probe-based NanoString method achieved superior gene expression quantification results when compared to RQ-PCR in archived FFPE samples. We believe that this newly developed technique is optimal for large-scale validation studies using total RNA isolated from archived, FFPE samples.

Correlation among nuclear localization of NuMA-RARα, deregulation of gene expression and leukemic phenotype of hCG-NuMA-RARα transgenic mice.

Acute promyelocytic leukemia (APL) is a model system of aberrant transcription in cancer. We sought to elucidate the mechanism of action of the variant fusion NuMA-RARα in APL, using the hCG-NuMA-RARα transgenic model. We report that subcellular localization of NuMA-RARα in transgenic mice is dependent upon its protein expression and transgene dosage. Subcellular localization of the fusion is inversely correlated with extent of gene deregulation at the mRNA level for Cebpα, Cebpɛ and Pu.1. Finally, we report that phenotype onset is correlated with NuMA-RARα copy number; mice with higher copy number developing disease later than those with lower copy number.

Myeloid leukemia with promyelocytic features in transgenic mice expressing hCG-NuMA-RARalpha.

Acute promyelocytic leukemia (APL) is characterized by the accumulation of abnormal promyelocytes in the bone marrow (BM), and by the presence of a reciprocal chromosomal translocation involving retinoic acid receptor alpha (RARalpha). To date, five RARalpha partner genes have been identified in APL. NuMA-RARalpha was identified in a pediatric case of APL carrying a translocation t(11;17)(q13;q21). Using a construct containing the NuMA-RARalpha fusion gene driven by the human cathepsin G promoter (hCG-NuMA-RARalpha), two transgenic mouse lines were generated. Transgenic mice were observed to have a genetic myeloproliferation (increased granulopoiesis in BM) at an early age, and rapidly developed a myeloproliferative disease-like myeloid leukemia. This leukemia was morphologically and immunophenotypically indistinguishable from human APL, with a penetrance of 100%. The phenotype of transgenic mice was consistent with a blockade of neutrophil differentiation. NuMA-RARalpha is therefore sufficient for disease development in this APL model.